Mapping the acylated proteome: a chemical genetic approach
Lead Research Organisation:
Imperial College London
Department Name: Chemistry
Abstract
With the completion of several important genome projects, such as the Human Genome Project, we now have the basic code describing the proteins made by cells which carry out the essential processes of life. However, this is only the beginning of our journey towards fully understanding how cells function: the focus of research has now moved to unravelling the role of each protein in a cell, and when and where it is carried out. To further complicate the story, the structure of a protein is frequently modified after its initial creation (its 'translation' from the genetic code) by other proteins called enzymes, a process termed 'post-translational modification'. These modifications can have critically important functions in the cell; for example, they are involved in our immune response to disease and the mechanism of infection by viruses, and defective modification is implicated in diseases such as cancer. Drugs which target the post-translational enzymes in pathogenic organisms such as fungi and parasites (for example, Plasmodium falciparum, the single-celled parasite which causes malaria) are under development, and could make a major impact on our ability to control these diseases. However, due to the complexity of the total set of thousands of different proteins in a cell (dubbed the 'proteome'), it has proven difficult to identify which proteins are modified, and what effect the modification has on a protein's function. Identifying the modified proteome is a key step towards understanding the role of post-translational modification, and to discover what effects (and side-effects) the drugs mentioned above may have in the cell. The proposed research aims to address this challenge by integrating recent discoveries in organic chemistry with cutting-edge whole-proteome analysis. We aim to exploit the cell's own enzymes to introduce a chemical 'tag' into proteins which are modified by a process termed 'acylation'. The tags are designed so as not to disrupt the cell's normal function, and we can very selectively 'capture' and purify the tagged acyl proteins with specially designed reagents. This will allow us to hugely increase the sensitivity of whole-proteome analysis towards acyl modifications, revealing hitherto unidentified acyl proteins and opening up new lines of investigation into the role of acylation in cells. Later, we intend to exploit this tag and capture methodology to analyse the effect of drugs on the modified proteome in single-celled parasites, and even to enable us to see modified proteins under a microscope as they move around the cell. This work will bring together chemists at Imperial College London and biologists at the University of York in an 'interdisciplinary' collaboration / that is, at the interface between the traditional disciplines of Chemistry and Biology.
Technical Summary
Co- or post-translational acylation of proteins is known to modulate myriad cellular processes including protein trafficking and the immune response, and the enzymes which perform post-translational acylation (acyl:protein transferases) play a key role in trafficking proteins between the membrane-delimited compartments of the cell. There is a pressing need to define the complete repertoire of myristoylated and palmitoylated proteins, respectively dubbed the 'myristome' and 'palmitome', and a generic method for their identification would enable and accelerate investigations into the functional biology of protein acylation. Tagging-by-substrate (TbS) is a powerful emerging technology that has the potential to overcome the problems commonly encountered in high-throughput proteomics of post-translationally modified proteins. In this approach, a synthetic transferase substrate bearing a biologically inert chemical tag (usually an azide or alkyne) is fed to cells, and incorporated into modified proteins metabolically in vivo. Tagged proteins are then captured from lysed cells in vitro using a highly selective bioorthogonal reaction, and by incorporating a dye or affinity label into the capture reagent the modified proteome can be selectively visualised or enriched, greatly enhancing high-throughput identification. This radically new approach to post-translational proteomics has already seen notable success for the labelling of certain glycosylated and farnesylated proteins in vivo. We intend to develop TbS for high-throughput myristomics and palmitomics, to provide a general cross-species method to enrich, visualise and identify acylated proteins in a wild-type cell-line. This work will generate invaluable data about the targets of acylation in vivo, present new targets for functional biology studies and greatly enhance the prediction of the myristome from genomic data by elucidating the sequence specificity of myristoylation.
Organisations
People |
ORCID iD |
Edward Tate (Principal Investigator) |
Publications
Goncalves V
(2012)
A fluorescence-based assay for N-myristoyltransferase activity.
in Analytical biochemistry
Broncel M
(2012)
A new chemical handle for protein AMPylation at the host-pathogen interface.
in Chembiochem : a European journal of chemical biology
Price H
(2012)
A role for the vesicle-associated tubulin binding protein ARL6 (BBS3) in flagellum extension in Trypanosoma brucei
in Biochimica et Biophysica Acta (BBA) - Molecular Cell Research
Kalesh KA
(2014)
A succinyl lysine-based photo-cross-linking peptide probe for Sirtuin 5.
in Organic & biomolecular chemistry
Heal WP
(2008)
Activity based chemical proteomics: profiling proteases as drug targets.
in Current drug discovery technologies
Heal WP
(2011)
Activity-based probes: discovering new biology and new drug targets.
in Chemical Society reviews
Serwa R
(2011)
Activity-based profiling for drug discovery.
in Chemistry & biology
Heal WP
(2012)
Application of activity-based protein profiling to the study of microbial pathogenesis.
in Topics in current chemistry
Alibhai D
(2013)
Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation.
in Journal of biophotonics
Kelly D
(2015)
Automated multiwell fluorescence lifetime imaging for Förster resonance energy transfer assays and high content analysis
in Analytical Methods
Tapodi A
(2019)
BFSP1 C-terminal domains released by post-translational processing events can alter significantly the calcium regulation of AQP0 water permeability
in Experimental Eye Research
Heal WP
(2011)
Bioorthogonal chemical tagging of protein cholesterylation in living cells.
in Chemical communications (Cambridge, England)
Thongyoo P
(2008)
Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides.
in Organic & biomolecular chemistry
Dang TH
(2010)
Chemical probes of surface layer biogenesis in Clostridium difficile.
in ACS chemical biology
Storck EM
(2013)
Chemical proteomics: a powerful tool for exploring protein lipidation.
in Biochemical Society transactions
Bradshaw RT
(2011)
Comparing experimental and computational alanine scanning techniques for probing a prototypical protein-protein interaction.
in Protein engineering, design & selection : PEDS
Douse CH
(2014)
Crystal structures of stapled and hydrogen bond surrogate peptides targeting a fully buried protein-helix interaction.
in ACS chemical biology
Rackham MD
(2014)
Design and synthesis of high affinity inhibitors of Plasmodium falciparum and Plasmodium vivax N-myristoyltransferases directed by ligand efficiency dependent lipophilicity (LELP).
in Journal of medicinal chemistry
Yu Z
(2012)
Design and synthesis of inhibitors of Plasmodium falciparum N-myristoyltransferase, a promising target for antimalarial drug discovery.
in Journal of medicinal chemistry
Rackham MD
(2013)
Discovery of novel and ligand-efficient inhibitors of Plasmodium falciparum and Plasmodium vivax N-myristoyltransferase.
in Journal of medicinal chemistry
Goncalves V
(2012)
Discovery of Plasmodium vivax N-myristoyltransferase inhibitors: screening, synthesis, and structural characterization of their binding mode.
in Journal of medicinal chemistry
Brannigan JA
(2014)
Diverse modes of binding in structures of Leishmania major N-myristoyltransferase with selective inhibitors.
in IUCrJ
Guttery DS
(2014)
Genome-wide functional analysis of Plasmodium protein phosphatases reveals key regulators of parasite development and differentiation.
in Cell host & microbe
Heal WP
(2010)
Getting a chemical handle on protein post-translational modification.
in Organic & biomolecular chemistry
Wright MH
(2015)
Global analysis of protein N-myristoylation and exploration of N-myristoyltransferase as a drug target in the neglected human pathogen Leishmania donovani.
in Chemistry & biology
Wright MH
(2016)
Global Profiling and Inhibition of Protein Lipidation in Vector and Host Stages of the Sleeping Sickness Parasite Trypanosoma brucei.
in ACS infectious diseases
Thinon E
(2014)
Global profiling of co- and post-translationally N-myristoylated proteomes in human cells.
in Nature communications
Tate EW
(2015)
Global profiling of protein lipidation using chemical proteomic technologies.
in Current opinion in chemical biology
Tate EW
(2011)
Highlights from the 46th EUCHEM Conference on stereochemistry, Bürgenstock, Switzerland, May 2011.
in Chemical communications (Cambridge, England)
Thongyoo P
(2007)
Immobilized protease-assisted synthesis of engineered cysteine-knot microproteins.
in Chembiochem : a European journal of chemical biology
Thomas JC
(2010)
Interaction and dynamics of the Plasmodium falciparum MTIP-MyoA complex, a key component of the invasion motor in the malaria parasite.
in Molecular bioSystems
So S
(2010)
Membrane enhanced peptide synthesis.
in Chemical communications (Cambridge, England)
Bowyer PW
(2007)
Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum.
in The Biochemical journal
Heal WP
(2011)
Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry.
in Nature protocols
Bradshaw R
(2012)
Mutational Locally Enhanced Sampling (MULES) for quantitative prediction of the effects of mutations at protein-protein interfaces
in Chemical Science
Heal WP
(2008)
N-Myristoyl transferase-mediated protein labelling in vivo.
in Organic & biomolecular chemistry
Tate EW
(2014)
N-Myristoyltransferase as a potential drug target in malaria and leishmaniasis.
in Parasitology
Brannigan JA
(2010)
N-myristoyltransferase from Leishmania donovani: structural and functional characterisation of a potential drug target for visceral leishmaniasis.
in Journal of molecular biology
Thinon E
(2016)
N-Myristoyltransferase Inhibition Induces ER-Stress, Cell Cycle Arrest, and Apoptosis in Cancer Cells.
in ACS chemical biology
Bowyer PW
(2008)
N-myristoyltransferase: a prospective drug target for protozoan parasites.
in ChemMedChem
Ciepla P
(2014)
New chemical probes targeting cholesterylation of Sonic Hedgehog in human cells and zebrafish.
in Chemical science
Tam Dang TH
(2012)
Novel inhibitors of surface layer processing in Clostridium difficile.
in Bioorganic & medicinal chemistry
Olaleye TO
(2014)
Peptidomimetic inhibitors of N-myristoyltransferase from human malaria and leishmaniasis parasites.
in Organic & biomolecular chemistry
Gray K
(2014)
Potent and specific inhibition of the biological activity of the type-II transmembrane serine protease matriptase by the cyclic microprotein MCoTI-II.
in Thrombosis and haemostasis
Thongyoo P
(2009)
Potent inhibitors of beta-tryptase and human leukocyte elastase based on the MCoTI-II scaffold.
in Journal of medicinal chemistry
Delmotte A
(2011)
Protein multi-scale organization through graph partitioning and robustness analysis: application to the myosin-myosin light chain interaction.
in Physical biology
Wright MH
(2010)
Protein myristoylation in health and disease.
in Journal of chemical biology
Berry AF
(2010)
Rapid multilabel detection of geranylgeranylated proteins by using bioorthogonal ligation chemistry.
in Chembiochem : a European journal of chemical biology
Description | Multiple chemical tools have been established that enable biology to be explored in new ways - for example, we can now unlock the biology and therapeutic potential of a wide range of protein modification pathways in cells of all types, from bacteria and viruses to parasites, human cells and living animals. |
Exploitation Route | We have used our findings to validate drug targets in malaria and cancer, and collaborated very widely with biologists from all fields of research, including bacteriologists, virologists, parasitologists, and human biology to understand how fundamental pathways function at a level of detail not previously possible. |
Sectors | Agriculture, Food and Drink,Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
URL | http://www.bbsrc.ac.uk/news/research-technologies/2013/131223-pr-proteomics-tool-new-malaria-drug.aspx |
Description | We have established the potential druggability of a range of pathways that were previously very difficult to address before our research was undertaken. |
First Year Of Impact | 2013 |
Sector | Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |